biotechnology using biology to solve societys problems
TRANSCRIPT
BiotechnologyBiotechnologyUsing Biology to Solve Society’s Using Biology to Solve Society’s
ProblemsProblems
13.1 What Is Biotechnology?13.1 What Is Biotechnology?
Genetic engineering – modify DNA to Genetic engineering – modify DNA to produce ______ (?)produce ______ (?) Recombinant DNA – genetically Recombinant DNA – genetically
modified organisms; recombinantmodified organisms; recombinant AgricultureAgriculture Medicines Medicines
Forensics Forensics
Resistant to fungus
13.2 Natural Recombination 13.2 Natural Recombination
Recombination of genes happens Recombination of genes happens naturally in sexually reproducing naturally in sexually reproducing organismsorganisms Meiosis and fertilizationMeiosis and fertilization
Recombination of genesRecombination of genes Law of SegregationLaw of Segregation Law of Independent AssortmentLaw of Independent Assortment Random chanceRandom chance
Crossing overCrossing over
Bacterial RecombinationBacterial Recombination
Bacteria – binary fission (asexual, Bacteria – binary fission (asexual, ‘cloning’)‘cloning’) No genetic variationNo genetic variation
Genetic recombination by 3 means:Genetic recombination by 3 means: Mutation – ‘goof’ in the codeMutation – ‘goof’ in the code Transformation – pick up DNA from Transformation – pick up DNA from
environmentenvironment Transduction – viruses transfer DNA Transduction – viruses transfer DNA Conjugation – bacteria ‘swap’ DNAConjugation – bacteria ‘swap’ DNA
TransformatioTransformationn
Bacteria can absorb DNA from Bacteria can absorb DNA from the environment the environment
Enables some bacteria to Enables some bacteria to become resistant to antibiotics become resistant to antibiotics (MRSA)(MRSA) Griffith’s experiment Griffith’s experiment Non-pathogen became pathogenNon-pathogen became pathogen
plasmid
bacterialchromosome
DNAfragments
bacterialchromosome
Transformation with DNA fragment
Bacterium
bacterialchromosome
Transformation with plasmid
plasmid
Transformation
ConjugationConjugation
Transfer of genes by bacteria that are Transfer of genes by bacteria that are temporarily ‘joined’ temporarily ‘joined’ E. coliE. coli
PiliPili - -‘appendage’ on the surface of a ‘appendage’ on the surface of a bacterium through which cytoplasm can bacterium through which cytoplasm can movemove
PlasmidsPlasmids
Circular DNA in bacteria that carries Circular DNA in bacteria that carries ‘extra’ genes‘extra’ genes F = ‘fertility’; the ability to grow sex piliF = ‘fertility’; the ability to grow sex pili R = various resistance factor(s)R = various resistance factor(s)
TransductiTransductionon
Transfer of genes from Transfer of genes from one bacterium to another one bacterium to another by phagesby phages Hershey-Chase; used T4 Hershey-Chase; used T4
phage to determine that phage to determine that DNA was the molecule of DNA was the molecule of heredityheredity
Restriction EnzymesRestriction Enzymes Bacteria have enzymes that cut DNA Bacteria have enzymes that cut DNA
in in specificspecific locations locations ‘‘Cut out’ phagesCut out’ phages
Enzymes have recognition sites Enzymes have recognition sites Specific sequence of nucleotidesSpecific sequence of nucleotides Palindromes Palindromes
Race car; anna; madam; flee to me Race car; anna; madam; flee to me remote elf; gnu dung; lager sir is remote elf; gnu dung; lager sir is regal; tuna nutregal; tuna nut
Restriction enzymes
BiotechnologyBiotechnology
ForensicsForensics
AgriculturalAgricultural
Medical Medical
Forensics – DNA Forensics – DNA FingerprintingFingerprinting
Restriction enzymes cut pieces Restriction enzymes cut pieces of DNA in very of DNA in very specificspecific locations locations Create pieces of DNA of Create pieces of DNA of
differing lengths differing lengths Restriction Fragment Length Restriction Fragment Length
Polymorphisms, FFLPs Polymorphisms, FFLPs
Other restriction enzymes
ForensicsForensics
RFLPsRFLPs
ForensicsForensics
Large and small pieces can be Large and small pieces can be separated separated
Gel electrophoresisGel electrophoresis – process used to – process used to separate RFLPsseparate RFLPs
1.1. Cut DNA with restriction enzymesCut DNA with restriction enzymes
2.2. DNA ‘loaded’ into a gel bed with wellsDNA ‘loaded’ into a gel bed with wells
3.3. Electricity ‘pulls’ the negative DNA towards Electricity ‘pulls’ the negative DNA towards the positive pole through the gel bedthe positive pole through the gel bed
4.4. Large pieces move slower creating Large pieces move slower creating separationseparation
Gel electrophoresis
DNA samples are pipetted into wells (shallow slots) in the gel. Electrical current is sent through the gel (negative at end with wells, positive at opposite end.)
gel
power supply
wells
pipetter
Electrical current moves DNA segments through the gel. Smaller pieces of DNA move farther toward the positive electrode.
DNA “bands”(not yet visible)
Gel electrophoresis
Southern Blotting TechniqueSouthern Blotting Technique
Too many bands are created during Too many bands are created during electrophoresiselectrophoresis
Have to make specific ‘bands’ visibleHave to make specific ‘bands’ visible Nitrocellulose paper is placed on the gelNitrocellulose paper is placed on the gel Alkaline solution unwinds the DNA helixAlkaline solution unwinds the DNA helix Separated strands of DNA are absorbed Separated strands of DNA are absorbed
by the nitrocellulose paper (blotting)by the nitrocellulose paper (blotting)PCR
Gel is placed on special nylon “paper.” Electrical current drives separatedDNA out of gel onto nylon.
gel
nylon paper
Southern BlottingSouthern Blotting
Radioactive or colored probes are Radioactive or colored probes are mixed with the ‘blot’mixed with the ‘blot’
Probes pair with their Probes pair with their complimentary STRcomplimentary STR
Probes can be seenProbes can be seen
Nylon paper with DNA is bathed in a solution of labeled DNA probes (red) that are complementary to specific DNA segments in the original DNA sample.
nylon paper
solution of DNA probes (red)
STR #1: probe base-pairs and binds
probe label(colored molecule)
STR #2: probe cannot base-pair; does not bind
Complementary DNA segments are labeled by probes (red bands).
DNA ForensicsDNA Forensics STR – short, tandem repeats of STR – short, tandem repeats of
nucleotides (4 or 5) nucleotides (4 or 5) AGATAGATAGATAGATAGATAGATAGATAGATAGATAGAT Common in non-coding regions of Common in non-coding regions of
DNA (introns) DNA (introns) Number of times it repeats is variableNumber of times it repeats is variable DNA fingerprint - use multiple STR’s DNA fingerprint - use multiple STR’s
(10-13) to determine identity(10-13) to determine identity
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
T
A
A
T
A
T
T
A
T
A
T
A
T
A
G
C
A
T
A
T
G
C
A
T
T
A
8 side-by-side (tandem) repeatsof the same 4-nucleotide sequence,
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
A
T
G
C
A
T
T
A
A
T
G
C
G
C
T
A
A
T
STR name
Penta D
CSF
D16
D16: an STR on chromosome 16
DNA samples from13 different people
D7
D13
D5
151413121110
98
Num
ber
of r
epea
ts
Variety of STR’s used to isolate one individual
DNA fingerprint
ForensicsForensics
Polymerase Chain Reaction - Polymerase Chain Reaction - amplifies DNAamplifies DNA PCR copies a specific DNA PCR copies a specific DNA
sequencesequence Need to make more copies of the Need to make more copies of the
DNADNADNA is degraded (old)DNA is degraded (old)Not a large enough sampleNot a large enough sample
Each PCR cycle doubles the number of copies of the DNA
1 2 3PCRcycles 4 etc.
1 2 4 8DNAcopies 16 etc.
DNAfragmentto beamplified
One PCR cycle
originalDNA
90 °C 50 °C 72 °C
1 HeatingseparatesDNA strands.
2 Cooling allowsprimers andDNA polymeraseto bind.
3 New DNA strands aresynthesized.
primers DNApolymerase
new DNAstrands
Primers – RNA sequences that tell DNA polymerase where to start copying
Primers + free nucleotides + DNA polymerase + heat = copies of DNA
Copies can be used for forensics, cloning, recombinations
Transgenic OrganismsTransgenic Organisms
Using biotechnology to rearrange Using biotechnology to rearrange genomesgenomes
Medical Medical AgriculturalAgricultural Bioethics Bioethics
Cut both with the same restriction enzyme.
Mix Bt gene and plasmid; add DNA ligase to seal DNA.
Transform Agrobacterium tumifaciens with recombinantplasmid
Infect plant cell with transgenic bacterium.
Insert Bt gene into plant chromosome.
DNA including Bt gene Ti Plasmid
Bt gene
plant cell
plantchromosomes A. tumifaciens
A. tumifaciensplasmids
bacterialchromosome
Cloning a gene
Agricultural Use Agricultural Use
Knowing genome of and organism Knowing genome of and organism enables us to ‘interfere’ with normal enables us to ‘interfere’ with normal protein productionprotein production Insert, remove genes Insert, remove genes
Sterile Insect TechniqueSterile Insect Technique Irradiate then releaseIrradiate then release Insert lethal gene – then releaseInsert lethal gene – then release
Silk worm – industrial strength, glow-Silk worm – industrial strength, glow-in-the-dark silkin-the-dark silk
Transgenic OrganismsTransgenic Organisms
Transgenic mosquitoesTransgenic mosquitoes Anopheles sp. carry malaria parasiteAnopheles sp. carry malaria parasite Insert genes so that they cannot Insert genes so that they cannot
The introduction of genes that impair The introduction of genes that impair PlasmodiumPlasmodium (Malaria parasite)(Malaria parasite) development into mosquito populations is a strategy being development into mosquito populations is a strategy being considered for malaria control. The effect of the transgene on considered for malaria control. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of mosquito fitness is a crucial parameter influencing the success of this approach. We have previously shown that anopheline this approach. We have previously shown that anopheline mosquitoes expressing the SM1 peptide in the midgut lumen mosquitoes expressing the SM1 peptide in the midgut lumen ((intestineintestine) are impaired for transmission of ) are impaired for transmission of Plasmodium bergheiPlasmodium berghei. . Moreover, the transgenic mosquitoes had no noticeable fitness load Moreover, the transgenic mosquitoes had no noticeable fitness load compared with nontransgenic mosquitoes when fed on noninfected compared with nontransgenic mosquitoes when fed on noninfected mice. Here we show that when fed on mice infected with mice. Here we show that when fed on mice infected with P. bergheiP. berghei, , these transgenic mosquitoes are more fit (higher fecundity and these transgenic mosquitoes are more fit (higher fecundity and lower mortality) than sibling nontransgenic mosquitoes. In cage lower mortality) than sibling nontransgenic mosquitoes. In cage experiments, transgenic mosquitoes gradually replaced experiments, transgenic mosquitoes gradually replaced nontransgenics when mosquitoes were maintained on mice infected nontransgenics when mosquitoes were maintained on mice infected with gametocyte-producing parasites (strain ANKA 2.34) but not with gametocyte-producing parasites (strain ANKA 2.34) but not when maintained on mice infected with gametocyte-deficient when maintained on mice infected with gametocyte-deficient parasites (strain ANKA 2.33). These findings suggest that when parasites (strain ANKA 2.33). These findings suggest that when feeding on feeding on PlasmodiumPlasmodium-infected blood, transgenic malaria-resistant -infected blood, transgenic malaria-resistant mosquitoes have a selective advantage over nontransgenic mosquitoes have a selective advantage over nontransgenic mosquitoes. This fitness advantage has important implications for mosquitoes. This fitness advantage has important implications for devising malaria control strategies by means of genetic modification devising malaria control strategies by means of genetic modification of mosquitoes. of mosquitoes.
Transgenic malaria-resistant mosquitoes have a fitness advantage when feeding on Plasmodium-infected blood
Mauro T. Marrelli, Chaoyang Li, Jason Rasgon, Marcelo Jacobs-Lorena
Dolly and her ewe
Transgenic OrganismsTransgenic Organisms
Restriction enzymes cut bacterial Restriction enzymes cut bacterial plasmid DNA (pDNA)plasmid DNA (pDNA)
Same restriction enzymes cut ‘genes Same restriction enzymes cut ‘genes of interest’ from another organismof interest’ from another organism
‘‘Genes of interest’ added to a Genes of interest’ added to a solution with pDNAsolution with pDNA
DNA ligase (‘glue’) is added to ‘glue’ DNA ligase (‘glue’) is added to ‘glue’ pieces togetherpieces together
Biotechnology In AgricultureBiotechnology In Agriculture
ManyMany Crops Are Genetically Crops Are Genetically ModifiedModified Tomatoes – slow ripening processTomatoes – slow ripening process Tomatoes – insect resistanceTomatoes – insect resistance
Bacillus thuringiensis – Bacillus thuringiensis – produces a natural produces a natural insecticideinsecticide
Desired genes are cloned (insecticide Desired genes are cloned (insecticide gene)gene)
Biotechnology in AgricultureBiotechnology in Agriculture
Transgenic plants are Transgenic plants are cloned cloned in vitroin vitro
Plants are bred together Plants are bred together creating 2creating 2ndnd generation generation plant with insect-plant with insect-resistance gene resistance gene
Plants produce Plants produce antibodies to fight antibodies to fight disease (?)disease (?)
Fish given growth hormones; GMT@
13.5 How Is Biotechnology Used for 13.5 How Is Biotechnology Used for Medical Diagnosis and Treatment?Medical Diagnosis and Treatment?
DNA Technology Can DNA Technology Can Be Used to Diagnose Be Used to Diagnose Inherited DisordersInherited Disorders Restriction Enzymes Restriction Enzymes
May Cut Different May Cut Different Alleles of a Gene at Alleles of a Gene at Different LocationsDifferent Locations
Diagnosing sickle-cell Diagnosing sickle-cell anemia with restriction anemia with restriction enzymes enzymes
Normal allele
Sickle cell
normalglobin allele
Mst II cuts a normal globin allele in 2 places, but cuts thesickle-cell allele in 1 place.
sickle-cellglobin allele
Mst II Mst IIMst II
DNA probe
Mst II Mst II
DNA probe
large
small
AA AS SSHomozygous
Heterozy
gote
Homozygous sickle-cell
Medical TreatmentMedical Treatment
DNA Technology DNA Technology Can Be Used to Can Be Used to Treat DiseaseTreat Disease Examples of Examples of
Currently Used Currently Used Products Produced Products Produced by Recombinant by Recombinant DNA Methods DNA Methods
Medical Diagnosis and TreatmentMedical Diagnosis and Treatment
Treat Cystic FibrosisTreat Cystic Fibrosis Cure Severe Combined Immune Cure Severe Combined Immune
DeficiencyDeficiency
parents with genetic disease
fertilized egg withdefective gene
embryo withgenetic defect
therapeuticgene
genetically correctedcell from culture
egg cellwithoutnucleus
genetically correctedclone of original embryo
healthy baby
baby withgenetic disorder
genetically correctedegg cell
treated culture
viralvector
amniocentesis
vagina
placenta
uterus
fetus
amnioticfluid
chorionic villi
chorionic villus sampling (by suction)
cells: sex determination,biochemical and enzymatic studies
cell culture: biochemical studies, chromosomal analysis, analysis using recombinant DNA methods
centrifugefluid: compositionanalysis
13.6 Ethical Issues of 13.6 Ethical Issues of BiotechnologyBiotechnology
Should Genetically Modified Organisms Should Genetically Modified Organisms Be Permitted in Agriculture?Be Permitted in Agriculture? Are Foods from GMOs Dangerous to Eat?Are Foods from GMOs Dangerous to Eat? Are GMOs Hazardous to the Environment?Are GMOs Hazardous to the Environment?
Should a Human Genome Be Changed by Should a Human Genome Be Changed by Biotechnology?Biotechnology?
Genetic screening
Genetic counseling HGP – the
code
HGP - ethics
HGP - disease